The long term objective of this proposal is to understand, at the molecular level, the mechanisms by which the biological macromolecules function. The approach to this objective is to characterize structurally the metal sites in metalloproteins. Motivation for this work lies in the observation that biological macromolecules often show selectivities and sensitivities which are unrivaled by conventional chemical systems. If one understands the correlation between macromolecular structure and biological function, it should be possible to rationally control the reactivity of the macromolecule. Structural information will be obtained using metal specific spectroscopic probes, with particular emphasis on x-ray absorption spectroscopy. The underlying principle is that by determining the structure of the metal active-site and by comparing the site-structure for different derivatives of the protein, it is possible to learn a substantial amount about the functioning of the protein. The proposed experiments involve two classes of metal-containing proteins: metalloproteins that mediate the reactivity of O2 and the reduced forms of O2, and metal binding proteins involved in heavy-metal homeostasis. A more thorough understanding of biological oxygen metabolism will be valuable in designing homeogeneous catalysts which mimic biological reactions. A more thorough understanding of the interactions between metal ion concentration and gene expression will have profound implications for drug design. In both cases, an understanding of the mechanisms by which the biological system function should eventually have numerous practical applications. These include for example, the rational design of inhibitors for specific metabolic processes and the selective modification of metalloproteins so as to alter their reactivity. The specific systems to be studied include the Mn/Ca/Cl cluster that carries out photosynthetic oxygen evolution, the Fe and Mn superoxide dismutases, phthalate dioxygenase, and galactose oxidase. Within the area of metal ion homeostasis, studies are proposed for a system that detoxifies organomercurials, a system that confers Cu resistance, and a novel Zn- binding protein that regulates RNA translation.